No white grass glycoside L, extracts including the compound, and use of both as antiviral drugs

Abstract

The present invention discloses the use of a known compound, lobatoside L, as an antiviral drug. It also discloses an ethanol extract of Herba Zygophyllae containing lobatoside L and a Herba Zygophyllae extract for antiviral purposes. The Herba Zygophyllae ethanol extract, Herba Zygophyllae extract, and Herba Zygophyllae extract provided herein all demonstrate antiviral activity against the H1N1 (A / Puerto Rico / 8 / 34; PR8) virus as determined by the CCK-8 assay.

Description

Technical Field

[0001] The present invention belongs to the field of antiviral drugs, and particularly relates to gentianin L, an extract comprising the compound, and uses of the two as antiviral drugs. Background Art

[0002] Actinostemmalobatum (Maxim.) Maxim., also known as Actinostemmalobatum, celestial ball grass, and white grass, is the whole herb, seeds, and leaves of Actinostemmalobatum (Maxim.) Maxim., a plant of the Cucurbitaceae family. It is found throughout northern and southern my country, as well as in Korea, Japan, the Soviet Union, India, and Indochina. Actinostemmalobatum is cold in nature, bitter in taste, and slightly toxic. It has diuretic, anti-edema, heat-clearing, detoxifying, and dampness-removing properties. It is primarily used to treat edema caused by nephritis, ascites, snake bites, and the early stages of malnutrition.

[0003] Patent (CN108888649A) discloses saponin extracts extracted from Zygophyllum sibiricum (but does not disclose the specific components and chemical structure of the molecules), and believes that such extracts have anti-tumor activity. Patent (CN102432666A) discloses a method for extracting and isolating a compound, kusalaside L, from Zygophyllum sibiricum and discloses the use of the compound in the preparation of anti-tumor drugs.

[0004] However, there is no report in the prior art on the application of extracts and active ingredients of Herba Zygophyllae in antiviral aspects. Summary of the Invention

[0005] In response to the above technical problems, the present invention proposes a compound for use in preparing an antiviral drug, wherein the compound has a chemical structure as shown in formula (1):

[0006]

[0007] The compound of formula (1) is kusnezanol L.

[0008] The present invention also provides an antiviral use of the Herba Zygophyllae extract, wherein the Herba Zygophyllae extract comprises a compound of formula (1).

[0009] The present invention also proposes an antiviral use of the Herba Zygophyllae extract, wherein the Herba Zygophyllae extract is a Herba Zygophyllae ethanol extract prepared by subjecting the Herba Zygophyllae raw material drug to reflux extraction with a 72-78% ethanol solvent.

[0010] The present invention also proposes an antiviral use of the Herba Zygophyllae extract, wherein the Herba Zygophyllae extract is prepared by subjecting the Herba Zygophyllae ethanol extract to polarity gradient extraction with organic solvents of different polarities.

[0011] The present invention also proposes an antiviral use of the Herba Zygophyllae extract, wherein the Herba Zygophyllae extract is prepared by subjecting the Herba Zygophyllae extract to separation and purification by high performance liquid chromatography.

[0012] The present invention also provides a preparation for preparing antiviral drugs, which comprises one or more of the above-mentioned Zygophyllum herb extracts and pharmaceutical excipients.

[0013] The ethanol extract of Herba Zygophyllae, the Herba Zygophyllae extract and the Herba Zygophyllae essence extract proposed by the present invention are tested by CCK-8 method and all have antiviral activity against H1N1 (A / Puerto Rico / 8 / 34; PR8) virus. DETAILED DESCRIPTION

[0014] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0015] The Herba Zygophyllae API used in the present invention includes fresh or dried Herba Zygophyllae whole plants, seeds, and leaves. A preferred embodiment uses dried raw materials.

[0016] Example 1 Preparation of Zygophyllum ethanol extract

[0017] Example 1-1

[0018] Take 1.0 kg of the whole herb of Herba Zygophyllae, extract it with 75% ethanol under reflux for 3 times, each time for at least 2 hours, combine the extracts, and recover the ethanol under reduced pressure to obtain 650 g of Herba Zygophyllae ethanol extract.

[0019] Example 1-2

[0020] Take 1.0 kg of the whole herb of Herba Zygophyllae, extract it with 72% ethanol under reflux for 3 times, each time for at least 2 hours, combine the extracts, and recover the ethanol under reduced pressure to obtain 665 g of Herba Zygophyllae ethanol extract.

[0021] Examples 1-3

[0022] Take 1.0 kg of the whole herb of Herba Zygophyllae, extract it with 78% ethanol under reflux for 3 times, each time for at least 2 hours, combine the extracts, and recover the ethanol under reduced pressure to obtain 675 g of Herba Zygophyllae ethanol extract.

[0023] The water content of the ethanol extract of Herba Zygophyllae in Example 1 is 20-25%.

[0024] Example 2 Preparation of Herba Zygophyllae Extract

[0025] The ethanol extract of Zygophyllum is prepared by the step of polar gradient extraction with organic solvents of different polarities. Specifically, the ethanol extract of Zygophyllum obtained in Example 1-1 is suspended in water and extracted three times with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence. The extracts of different polarities are combined and the corresponding solvents are recovered to obtain 20g of Zygophyllum extract.

[0026] Example 3: Zygophyllum herb extract

[0027] The Herba Zygophylla extract is prepared by the steps of separating and purifying the Herba Zygophylla extract by high performance liquid chromatography. Specifically, 20 g of the Herba Zygophylla extract prepared in Example 2 is mixed and subjected to silica gel column chromatography, and eluted with dichloromethane-methanol-water (7:1:1-7:5:1) to obtain fractions 1-6; fraction 6 is separated by silica gel column chromatography and eluted with dichloromethane-methanol (2:1) to obtain 6.3; fraction 6.3 is separated by gel chromatography to obtain 6.3.1 and fraction 6.3.2, and fraction 6.3.2 is again subjected to high performance liquid chromatography to obtain the separated and purified Herba Zygophylla extract.

[0028] The chemical structure of the obtained Herba Zygophyllae extract was determined below.

[0029] First, it was found that it was recrystallized in methanol solvent to form a white powder, mp.320-322℃, [α] 20 D —76° (c 0.25, MeOH). 10% sulfuric acid ethanol solution turns blue, and the Liebermann-Burchard reaction and the Molish reaction are positive. The above phenomena suggest that it may be a triterpenoid saponin compound. Glucose, arabinose, and rhamnose were detected by hydrolysis with 10% sulfuric acid. HR-ESI-MS gave: m / z 1417.6249 [M+Na]+ quasi-molecular ion peak (C65H102O32Na+, calculated value 1417.6252). Combined with NMR data, its molecular formula can be inferred to be C65H102O32. Strong absorption peaks at 3442cm-1 and 1734cm-1 in the infrared spectrum indicate the presence of hydroxyl and carbonyl groups in the structure.

[0030] The 1H-NMR (600MHz, C5D5N) spectrum gives 8 methyl proton signals in the structure as 0.98 (3H, s), 1.02 (3H, s), 1.32 (3H, s), 1.44 (3H, d, J = 6.0 Hz), 1.52 (3H, s), 1.69 (3H, s), 1.88 (3H, s), 1.92 (3H, s); 1 olefinic hydrogen proton signal δ5.64 (1H, br.s); 5 sugar end proton signals δ5.12 (1H, d, J = 7.8 Hz), 5.29 (1H, d, J = 7.2 Hz), 5.98 (1H, d, J = 7.2 Hz), 6.22 (1H, br.s) and 5.20 (1H, d, J = 7.8 Hz). According to the splitting of methyl group and 13C-NMR data analysis, it is inferred that the extract of Herba Zygophyllae is oleanane-type pentacyclic triterpenoid saponin.

[0031] The 13C-NMR (150 MHz, C5D5N) spectrum gave a total of 65 carbon signals, of which 30 carbons were carbon signals on the oleanane core structure, 29 carbons were carbon signals on sugars, and 6 carbons were 3-hydroxy-3-methylglutaric acid ester signals. Of the 30 carbon signals in the core structure, there are six methyl carbon signals at δ15.4 (C-24), 17.6 (C-25), 17.8 (C-26), 27.4 (C-27), 33.2 (C-29), and 24.4 (C-30); four oxygen-linked carbon signals at 70.2 (C-2), 83.1 (C-3), 73.5 (C-16), and 64.5 (C-23); two olefinic carbon signals at δ123.4 (C-12) and 145.3 (C-13); and one ester carbonyl signal at δ176.4 (C-28). Comparison of these data with those reported for polygalacic acid in the literature by 1H-NMR reveals that, with the exception of differences in chemical shifts at positions 2, 3, and 28, the remaining data are generally consistent. Therefore, the molecular structure of the extract of Zygophyllum sibiricum was determined to be polygalacic acid, and the glycosyl substitutions at positions 3 and 28 were determined based on the glycosylation displacement pattern. Among the 29 sugar signals, 23 were linked to oxygen at δ84.2, 78.9, 78.0, 78.0, 77.9, 77.8, 77.3, 77.3, 77.3, 75.9, 75.0, 74.9, 73.5, 72.2, 71.6, 71.1, 71.0, 69.5, 68.1, 67.5, 65.1, 62.7, and 62.5; 5 were linked to di-linked oxygen at δ105.8, 105.6, 103.8, 102.6, and 94.7; and 1 was a methyl at δ18.5, which was the carbon 6 signal of rhamnose. Combining the corresponding sugar terminal proton signals in 1H-NMR with the results of acid hydrolysis, it can be determined that there are three glucoses, one rhamnose and one arabinose in the extract of the zygote. The three groups of glucose carbon signals are Glc: δ103.8, 84.2, 77.3, 71.6, 78.0, 62.7; Glc': δ105.6, 77.3, 78.0, 71.1, 75.9, 65.1; Glc": δ105.8, 74.9, 77.9, 71.0, 77.8, 62.5; the group of rhamnose carbon signals are δ102.6, 72.2, 78 .9, 73.5, 68.1, 18.5; a group of carbon signals of arabinose are δ94.7, 77.3, 75.0, 69.5, 67.5. The six carbon signals of 3-hydroxy-3-methylglutarate group are δ171.4 (C-1'), 47.1 (C-2'), 70.3 (C-3'), 47.1 (C-4'), 171.9 (C-5'), 25.9 (C-6').Comparison of the carbon spectrum data from the extract of the herb with that of the compound tubeimoside III reported in the literature revealed that, with the exception of the outer xylose, the data were essentially identical. Therefore, it was determined that the inner sugar linkage of this compound was consistent with that of tubeimoside III, with the difference being that the outer xylose was replaced with glucose.

[0032] In the HMBC spectrum, the nomonic signal of glucose δ5.12 (1H, d, J = 7.8 Hz, Glc H-1) has a long-range correlation with the 3-position δ83.1 (C-3) signal of the parent nucleus, and the nomonic signal of Glc' δ5.29 (1H, d, J = 7.2 Hz, Glc'H-1) has a long-range correlation with the 2-carbon signal of Glc δ84.2 (GlcC-2), indicating that Glc is connected to the C-3 position of the parent nucleus and Glc' is connected to the 2 position of Glc; the nomonic signal of arabinose δ5.98 (1H, d, J = 7.2 Hz, AraH-1) has a long-range correlation with the C-3 position of the parent nucleus. The signal of δ176.4 at position C-28 has a long-range correlation, indicating that Ara is connected to the C-28 position of the parent nucleus; the signal of δ6.22 (1H, br.s, RhaH-1) of the terminal proton of Rha has a long-range correlation with the signal of δ77.3 (AraC-2) at position 2 of Ara, indicating that Rha is connected to the AraC-2 position; the signal of δ5.20 (1H, d, J=7.8 Hz, Glc”H-1) of the terminal proton of Glc” has a long-range correlation with the signal of δ78.9 (Rh The 6-position proton signal of Glc' δ5.02 (1H, d, J=11.4 Hz, Glc'H-6) has a long-range correlation with the 1-position carbon signal of hydroxymethylglutaryl group δ171.4 (C-1'), and the 4-position proton signal of Rha δ6.05 (1H, t, J=9.6 Hz, RhaH-4) has a long-range correlation with the 5'-position carbon signal of hydroxymethylglutaryl group δ171.9 (C-5'). The results indicate that the two ends of the 3-hydroxy-3-methylglutarate group (positions 1' and 5') are attached to positions 6 and 4 of Glc' and Rha, respectively. The absolute configuration of the sugar can be determined by gas chromatography. Acid hydrolysis and derivatization of the compound, followed by comparison of its gas chromatogram with that of standard sugar derivatives, revealed D-glucose, L-rhamnose, and L-arabinose, respectively. Based on the chemical shift values ​​and coupling constants of the terminal hydrogens, the compounds were identified as β-D-glucose, α-L-rhamnose, and α-L-arabinose, respectively.

[0033] In summary, the structure of the compound was identified as lobatoside L, and the structure was formula (1):

[0034]

[0035] Test Example 1 Anti-virus test

[0036] The CCK-8 assay was used to determine the activity of the following test substances against the H1N1 (A / Puerto Rico / 8 / 34; PR8) virus.

[0037] Test samples: the ethanol extracts of Herba Zygophyllae prepared in Examples 1-1, 1-2, and 1-3, the Herba Zygophyllae extract prepared in Example 2, the compound obtained in Example 3, and acyclovir as a positive control were prepared in phosphate buffer to prepare four groups of test samples containing cyperus globulin concentration gradients of 1.25, 2.5, 5, 10, and 20 μmol / L, respectively.

[0038] Test method:

[0039] Step (1)

[0040] The virus host cells (A549) were stored in RPMI1640 medium, supplemented with 100 μg / mL penicillin and streptomycin and 10% fetal bovine serum, and cultured in a cell incubator at 37°C and 5% CO2. After the cells adhered to the wall, the old medium was removed, the cells in the medium were washed with phosphate buffer, and an appropriate amount of trypsin was added to digest the cells. When the intercellular gaps increased and the cytoplasm retracted, the digestion was stopped, the digestion fluid was aspirated, and the cells were washed with phosphate buffer. The culture medium was added and the cells were transferred to a centrifuge tube for centrifugation. The supernatant was removed and fresh culture medium was added to form a cell suspension. 5×10 cells were seeded per well of a 96-well plate. 4 Cells (100 μL culture medium) were cultured for 24 h, and the viruses were infected on A549 cells in good growth state, added to the culture medium containing 10% fetal bovine serum, and cultured in a cell incubator at 37°C and 5% CO2 for 24 h. The culture of virus cells was stopped when the pathological effect reached 90%, and the virus amplification was completed.

[0041] Step (2)

[0042] 100 μL of the above test samples were added to each well of a 96-well plate. Three replicate wells were set for each concentration of each test sample. Then, 100 times the viral suspension at half the cell infection dose was added and cultured at 37°C and 5% CO2 for 4 h. When about 80% of the cells became cytopathic, the supernatant was discarded and 100 μL of 5 mg / mL CCK-8 solution was added. The cells were cultured in a cell incubator for 2 h, the supernatant was discarded, 100 μL of DMSO was added and shaken. The A value of each well was measured at a wavelength of 530 nm using a microplate reader to calculate the virus inhibition rate and IC 50 .

[0043] Table 1 Anti-virus test results

[0044]

[0045] The test results show that the extracts of Herba Zygophyllae of Examples 1-1, 1-2, and 1-3 showed an inhibitory effect on the H1N1 virus when the concentration of cyperus glycoside L was 150 μmol / L; the n-butanol extract of Herba Zygophyllae of Example 2 showed an inhibitory effect on the H1N1 virus when the concentration of cyperus glycoside L was 50 μmol / L; the compound isolated and extracted in Example 3 (without cyperus glycoside L) showed an inhibitory effect on the H1N1 virus at a concentration of 2.5 μmol / L, which was equivalent to the antiviral effect of the positive control acyclovir.

[0046] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A compound for use in preparing an antiviral drug, characterized in that: The compound has a chemical structure as shown in formula (1): Formula (1); The antiviral drug is a drug against H1N1 virus.

Images